JP4905624B2 - Combination therapy of peptide vaccine administration and estramustine treatment - Google Patents

Description

Detailed Description of the Invention

[Technical field]
The present invention relates to immunotherapy for cancer, specifically the treatment of hormone refractory prostate cancer.

[Background technology]
Optimal treatment of patients with metastatic hormone refractory prostate cancer (HRPC) continues to be a typical goal of cancer research. The median survival of patients with metastatic HRPC is about 12 months [13]. Chemotherapy with mitoxantrone shows a transient improvement [1,2], but no treatment has yet been demonstrated to prolong survival. Recently, in a phase II clinical trial of estramustine or taxane-based therapy, it was reported that serum prostate specific antigen (PSA) levels were reduced by ≧ 50% in 45% to 67% of patients [48]. . However, these combinations cannot be used for patients with poor performance status because they involve significant nausea, diarrhea, leukopenia, and cumulative fluid retention and increase the risk of thrombotic events. In addition, none of these treatments have extended survival and the number of patients in this study is limited.

  Numerous tumor antigens recognized by human leukocyte associated antigen (HLA) class I-restricted cytotoxic T lymphocytes (CTLs) have been identified in the last decade [9, 10] and a new treatment for HRPC using tumor vaccines has been developed. Have been studied. A phase I / II clinical trial of dendritic cell-based immunotherapy was conducted, and vaccines containing recombinant prostate specific membrane antigen (PSMA) and adjuvant were also tested in prostate cancer patients [11, 12]. Our immunotherapy approach for HRPC patients, as reported previously [13, 14], is the use of peptide-specific CTL precursors in the circulating blood of cancer patients who respond to 30 vaccine candidates capable of inducing CTLs. Following the measurement prior to vaccine administration, only reactive peptides are administered (patient-directed peptide vaccine administration). We recently completed a phase I clinical trial for HRPC and evaluated the safe administration of these peptides [15]. Although the adverse events of this immunotherapy were milder than those of conventional treatments, the clinical response of this trial was limited. It is suggested that an additive anti-tumor effect could be achieved by the combined use of peptide vaccines and cytotoxic agents when the suppression of the immune system by cytotoxic agents is minimal.

  Estramustine phosphate is a stable conjugate of estradiol and nitrogen mustard that is antimitotic and causes disruption of microtubule formation [16]. Estramustine phosphate has been undergoing a number of Phase II and Phase III clinical trials as a secondary treatment as well as a primary treatment for HRPC over the past 25 years. The advantage of estramustine phosphate compared to other cytotoxic drugs is that it is easy to administer (oral administration) and is relatively well tolerated at effective doses.

  Although the combination of immunotherapy and cytotoxic drugs is not a new concept [27], the negative interactions that can occur due to the myelosuppressive nature of many cytotoxic drugs have attracted major attention. Cytotoxic drugs preferentially kill dividing cells as a feature of the activated immune system and may therefore inhibit the immune response. However, myelosuppression as the toxicity of estramustine phosphate has been rarely reported in patients treated with HRPC [16]. In a phase III clinical trial comparing the combination of estramustine phosphate and vinblastine with vinblastine alone, the combination method had a lower neutropenia rate than the single method (grades 2, 3, 4: 7%) 27%, 18% and 9% for 1% and 1%) [3].

[Disclosure of the Invention]
The purpose of the present invention is to determine the clinical and immunological response of HRPC patients to a combination of patient-directed peptide vaccination and oral estramustine phosphate, serum PSA, bone metabolism markers and clinical bone scan It is to analyze and evaluate the recording, peptide-specific CTL precursor by INF-γ release assay, and peptide-reactive IgG by enzyme-linked immunosorbent assay.

That is, the present invention relates to the following items:
(1) A method for treating prostate cancer, comprising administering to a patient in need thereof a therapeutically effective amount of a cancer antigen peptide-related substance and a low dose of estramustine or a salt thereof;
(2) The method according to (1), wherein the dose of estramustine or a salt thereof at a low dose is 140 to 560 mg / day;
(3) The method according to (1) or (2), wherein the cancer antigen peptide-related substance is patient-oriented;
(4) The method according to (3), wherein the cancer antigen peptide-related substance is selected from the group consisting of a cancer antigen protein, its cancer antigen peptide, its gene and derivatives thereof;
(5) The method according to any one of (1) to (4), wherein the cancer is hormone refractory prostate cancer;
(6) A pharmaceutical composition for treating prostate cancer, which is administered together with a cancer antigen peptide-related substance, comprising a low dose of estramustine or a salt thereof;
(7) The dose of low dose estramustine or a salt thereof is 140 to 560 mg / day,
(6) pharmaceutical composition;
(8) The pharmaceutical composition according to (6) or (7), wherein the cancer antigen peptide-related substance is patient-oriented;
(9) The pharmaceutical composition of (8), wherein the cancer antigen peptide-related substance is selected from the group consisting of a cancer antigen protein, its cancer antigen peptide, its gene and derivatives thereof;
(10) The pharmaceutical composition according to any of (6) to (9), wherein the cancer is hormone refractory prostate cancer; and (11) Estramustine for producing a medicament for treating prostate cancer Or use of a salt thereof, wherein the estramustine or a salt thereof is in a low dose and the medicament is administered together with a cancer antigen peptide-related substance.

[Best Mode for Carrying Out the Invention]
In accordance with the present invention, we have provided peptide vaccination and estramustine phosphate to human leukocyte antigen (HLA) -A24 or A2 positive metastatic hormone refractory prostate cancer (HRPC) patients who did not respond to prior peptide vaccine administration. A salt treatment combination therapy was administered, and as a result, this combination therapy was found to successfully enhance peptide-specific CTL precursors or peptide-specific IgG and reduce patient serum PSA levels.

  In a first aspect, the present invention is a method for treating prostate cancer comprising administering a therapeutically effective amount of a cancer antigen peptide-related substance and a low dose of estramustine or a salt thereof to a patient in need thereof Providing a method comprising:

  Prostate cancers treated by the present invention include hormone-responsive prostate cancer, hormone-refractory prostate cancer, and hormone-insensitive prostate cancer. including. The present invention preferably treats or prevents hormone refractory prostate cancer, particularly metastatic hormone refractory prostate cancer.

  Estramustine belongs to a group of medicines called antineoplastic drugs and is used worldwide for the treatment of several cases of prostate cancer. Estramustine is a combination of estradiol and nitrogen mustard. In the present invention, estramustine can be administered orally to the patient in the form of a salt, preferably a phosphate.

  The term “low dose” as used with respect to estramustine means an estramustine dose that is lower than the normal dose, specifically 140-560 mg / day, preferably 210-490 mg / day, more preferably 210 -420 mg / day, more preferably 280 mg / day. Low doses include, among others, 100-150 mg / day, 100-200 mg / day, 100-250 mg / day, 120-170 mg / day, 120-220 mg / day, 120-270 mg / day, 140-190 mg / day, 140 -240 mg / day, 140-290 mg / day, 160-210 mg / day, 160-260 mg / day, 180-230 mg / day and 180-280 mg / day.

  As used herein, the term “cancer antigen peptide-related substance” means a tumor antigen protein and its gene, a tumor antigen peptide derived from a tumor antigen protein and its gene, and derivatives thereof. A tumor antigen peptide is produced by degradation of a tumor antigen protein, which is a protein specific to a tumor, by intracellular proteasome and synthesized in the cell. The thus produced tumor antigen peptide binds to the MHC class I antigen (HLA antigen) in the endoplasmic reticulum to form a complex, and this complex is transported to the cell surface and presented as an antigen. The

  Tumor antigen proteins used in the present invention include the following: a protein named MAGE derived from human melanoma cells (Science, 254: 1643, 1991); a melanosomal protein, such as a melanocyte-specific tissue protein , Gp100 (J. Exp. Med., 179: 1005, 1994), MART-1 (Proc. Natl. Acad. Sci. USA, 91: 3515, 1994), and tyrosinase (J. Exp. Med., 178: 489, 1993); MAGE related protein (J. Exp. Med., 179: 921, 1994); β-catenin with tumor-specific amino acid mutation (J. Exp. Med., 183: 1185, 1996) ); And CDK4 (Science, 269: 1281, 1995); HER2-neu (J. Exp. Med., 181: 2109, 1995), p53 (mutant) (Proc. Natl. Acad. Sci. USA, 93: 14704, 1996); tumor markers such as CEA (J. Natl. Cancer Inst., 87: 982, 1995), PSA (J. Natl. Cancer Inst. , 89: 293, 1997); and viral proteins such as HPV (J. Immunol., 154: 5934, 1995) and EBV (Int. Immunol., 7: 653, 1995). Detailed descriptions of these substances are described in published reviews (eg, Immunol. Today, 18: 267, 1997; J. Exp. Med., 183: 725, 1996; and Curr. Opin. Immunol., 8: 628, 1996).

  Typical examples of tumor antigen peptides for use in the present invention include, but are not limited to, the following: tumor antigen peptides as described in WO97 / 46676, WO99 / 29715 and WO99 / 33977; B) derived tumor antigen peptide (WO99 / 67288); SART-1 derived tumor antigen peptide (WO00 / 06595); SART-3 derived tumor antigen peptide (WO00 / 12701); ART-1 derived tumor antigen peptide ( WO00 / 32770); tumor antigen peptide derived from SART-2 (J. Immunol, 164: 2565, 2000); tumor antigen peptide derived from lck (Eur. J. Immunol., 31: 323, 2001); derived from ART-4 Tumor antigen peptides (Cancer Res., 60: 3550, 2000); and ppMAPkk, WHSC2, UBE2V, HNRPL, EIF derived tumor antigen peptides (Cancer Res., 61: 2038, 2001).

  The gene for cancer antigen protein and cancer antigen peptide used in the present invention is prepared according to well-known methods such as the method described in Molecular Cloning 2nd Edt. Cold Spring Harbor Laboratory Press (1989) with reference to the above-mentioned literature. be able to.

  The cancer antigen protein, cancer antigen peptide and gene derivatives thereof used in the present invention mean artificial proteins and peptides and genes thereof prepared based on the amino acid sequences of the cancer antigen protein and peptide. Typical examples of derivatives have amino acid sequences in which a few amino acid residues are substituted, deleted and / or added to the amino acid sequences of natural cancer antigen proteins and peptides, and the natural cancer Proteins and peptides having an immune response inducing activity equivalent to that of antigenic proteins and peptides are included.

  According to the present invention, a patient-oriented cancer antigen peptide-related substance is administered. For example, to select a patient-oriented cancer antigen peptide, a vaccine candidate containing the antigen peptide is administered to a cancer patient, and the ability of the vaccine candidate to induce CTL is measured by conventional methods [13, 14]. This cancer antigen peptide-related substance is usually administered subcutaneously.

  In a second aspect, the present invention provides a pharmaceutical composition for treating prostate cancer, comprising a low dose of estramustine or a salt thereof and administered together with a cancer antigen peptide-related substance I will provide a. In another aspect, the present invention provides a pharmaceutical composition for treating prostate cancer, comprising a low dose of estramustine or a salt thereof and increasing a CTL precursor specific for a cancer antigen peptide-related substance. And / or a pharmaceutical composition that increases immunoglobulin G specific to a cancer antigen peptide-related substance. The meanings of the terms “prostate cancer”, “estramustine”, “low dose”, “cancer antigen peptide-related substance” have been described above.

[Example]
The present invention is further illustrated by the following examples, which are not intended to limit the invention in any way.

  Abbreviations used in the examples refer to the following terms: cytotoxic T lymphocytes (CTL); peripheral blood mononuclear cells (PBMC); human leukocyte antigen (HLA); hormone refractory prostate cancer (HRPC); prostate Specific antigen (PSA); bone metabolism marker (pyridinoline cross-linked type I collagen carboxy terminal telopeptide: ICTP); enzyme-linked immunosorbent assay (ELISA); delayed hypersensitivity (DHT); computed tomography (CT); complete response (CR); partial response (PR); disease progression (PD); and severe response (Ar).

Example 1
Patients and methods Patients Between February 2001 and September 2002, 20 human leukocyte antigen (HLA) A24 or A2 positive metastatic HRPC patients participated in a phase I study, and they were peptide specific. Cytotoxic cytotoxic T lymphocyte (CTL) precursor directed vaccine was administered [15]. Thirteen patients participated in a combination therapy of peptide vaccine administration and estramustine phosphate treatment if the condition worsened after at least three peptide vaccine administrations in a phase I study. Disease progression was defined by at least one of three criteria: ie, at least 2 consecutive weeks with at least 2 weeks increasing PSA levels by more than 25% from baseline; 2D measurable soft tissue Metastasis increased by more than 25%; and new lesions appeared on radionuclide bone scans. Serum PSA levels were measured using a tandem-R (Hybritech Inc., San Diego, CA) test with a normal range of 0-4.0 ng / mL. Other qualifications include Eastern Cooperative Oncology Group performance status 0 or 1, age 79 or less, granulocyte count 3000 / mm ³ or higher, hemoglobin 10 g / dl As described above, platelets of 100,000 / mm ³ or more, bilirubin was equal to or less than the normal limit set by the institution, and creatinine of 1.4 mg / dL or less were included. A negative serum test for hepatitis B and C was essential. Patients with severe illness or active secondary malignancy within 5 years were excluded from study participation. Exclusion criteria included those with signs of immunosuppression or autoimmune disease. All patients were explained and approved in accordance with institutional guidelines. This study was approved by the Kurume University School of Medicine Ethics Committee.

  Two of the 13 patients were excluded from immunological and clinical evaluation because the intended course of treatment was not completed (6 doses of vaccine) and there were no samples for immunological analysis. Therefore, 11 patients were eligible for immunological and clinical evaluation. The baseline characteristics of 11 patients treated with combination therapy are summarized in Table 1 below.

  At the time of enrollment in the study, the median% PABS for patients with bone metastases was 6.0 (range: 1.5-8.4). Prior treatment included hormone therapy (11 patients), radiation therapy for bone metastases (2 patients) and chemotherapy with estramustine phosphate (8 patients: 5 patients with estramustine alone; patients Three patients included estramustine + etoposide combination treatment). All 11 patients received peptide vaccine administration 3 times or more (median 6 times, range 3 to 23 times) before the start of combination therapy. The median duration of combination therapy was 13 months (range 6-21 months).

Patient-Directed Peptide Vaccine Administration Our immunotherapy approach for HRPC patients, as previously reported [13-15], was the 30 vaccine candidates with the ability to induce CTLs (14 peptides and HLA for HLA-A24 positive patients). -Peptide-specific CTL precursors in circulating blood of cancer patients that respond to A2 positive patients) are measured prior to vaccine administration, followed by administration of only reactive peptides (CTL precursors) Directed peptide vaccine). The peptides used in this study are listed in Table 2 below. These peptides were prepared by a multiple peptide system (Multiple Peptide System, San Diego, Calif.) Under standard conditions for pharmaceutical production control and quality control. All of these peptides have the ability to induce HLA-A24 or HLA-A2 restricted and tumor-specific CTL activity in peripheral blood mononuclear cells (PBMC) of cancer patients [17-23]. Thirty milliliters of peripheral blood was collected before the first vaccine administration and on the seventh day after every six vaccine administrations, and PBMCs were separated by Ficoll-Conlay density gradient centrifugation. Peptide-specific CTL precursors in PBMC were detected using a previously reported culture method [24]. Briefly, PBMC (1 × 10 ⁵ cells / well) were incubated in 200 μL culture medium with 10 μM of each peptide on a U-bottom 96-well microculture plate (Nunc, Roskilde, Denmark). This culture medium is 45% RPMI 1640 medium, 45% AIM-V ^™ medium (Invitrogen Corp., Carlsbad, Calif.), 10% FCS, 100 U / mL-interleukin 2 (IL-2) and 0.1 mM-MEM non- Contains essential amino acid solution (Invitrogen Corp.). Half of the medium was removed every 3 days up to 12 days and replaced with fresh medium containing the corresponding peptide (20 μM). 24 hours after the last stimulation on day 12 of culture, cells were harvested and washed 3 times, then C1R-A2402 pre-loaded with either the corresponding peptide or HIV peptide (RYLRQQLLGI) as a negative control or The ability to produce INF-γ in response to T2 cells was tested in HLA-A24 or HLA-A2PBMC, respectively. Target cells (C1R-A2402 or T2, 1 × 10 ⁴ / well) were pulsed with each peptide (10 μM) for 2 hours, then effector cells (1 × 10 ⁵ / well) were added to each well to a final volume of 200 μL. After 18 hours of incubation, the supernatant (100 μL) was collected, and the amount of INF-γ was measured using enzyme-linked immunosorbent assay (ELISA) (detection limit: 10 pg / mL). All experiments were performed twice in 4 different wells. For CTL precursors that respond to 30 peptides (14 peptides for HLA-A24 positive patients and 16 peptides for HLA-A2 positive patients), pre-vaccine PBMC were screened twice in each well in different wells. did. The results of each well were classified into 4 groups according to the p-value (both measurements Student's t-test), and the amount of INF-γ (response average for the corresponding peptide-response average for the HIV peptide) was as follows:
High response (Ar): p ≦ 0.1 and 500 ≦ net production;
A: moderate response (A): p ≦ 0.05 and 50 ≦ pure production;
B: p ≦ 0.05 and 25 ≦ pure production <50;
C: 0.05 <p ≦ 0.1 and 50 ≦ pure production.
Peptides were selected in the above order based on the evaluation of all 4 wells.

Combination therapy The peptide vaccine regimen was as follows: In the skin study, up to four 10 μg of each of the selected peptides were injected intradermally separately with a tuberculin syringe fitted with a 27 gauge needle. Immediate and delayed hypersensitivity (DHT) responses were measured 20 minutes and 24 hours after skin testing. Erythema and induration with diameters> 30 mm were defined as positive skin test reactions, and saline was used as a negative control for hypersensitivity assessment. Peptides were injected when immediate hypersensitivity was negative. Prior to the combination therapy, 3 mg / mL of each peptide was injected subcutaneously in the lateral thigh of each patient at a 2-week interval for a total of 6 injections. During combination therapy, 1 mg / mL of each peptide was injected at 4-6 week intervals.

  In the first two cases, estramustine phosphate was orally administered in two 140 mg capsules twice daily for a total daily dose of 560 mg. Severe immunosuppression was observed in these patients. To avoid severe immunosuppression, estramustine phosphate was reduced to 280 mg / day in the remaining 11 cases.

Immunological monitoring To assess immune responses during combination therapy, serum concentrations of peptide-specific CTL precursors and peptide-specific antibodies in PBMC were measured every 6 doses of vaccine. Peptide-specific CTL precursors in PBMC were detected using previously reported culture methods [24], and serum IgG levels specific for the administered peptide were detected using ELISA as previously reported [13- 15]. In addition, a new monitoring was performed to carefully measure estramustine-induced immunosuppression. That is, PBMCs were collected every 2 weeks, and 10 μM phytohemagglutinin (PHA), Epstein-Bar virus (EBV) -derived peptide having 10 ng / mL of HLA-A24 or -A2 binding motif and vaccine administration were performed in triplicate. The two different peptides were cultured for 2 days with 10 ng / mL (10 ⁴ cells / well). After culturing for 2 days, the amount of INF-γ in the cell-free supernatant was measured by three repeated tests, and the number of viable cells was also counted. To avoid bias in each assay, all PBMCs were cryopreserved once and four different PBMCs (two from healthy individuals, one from patients PBMC collected two weeks prior to the most recent vaccine administration, and one more recent) From one vaccine administration) were thawed simultaneously on the morning of the experiment. Responses to PHA, EBV-peptide and vaccinated peptide (INF-γ production) are thought to be mediated by resting T cells, memory T cells and activated T cells, respectively.

Clinical monitoring Patients were clinically monitored to assess the effects of combination therapy. Specifically, patients who participated in the study were monitored until disease death or intolerance, or consent was withdrawn. Clinical and laboratory evaluations were performed at each visit, and patients were informed of adverse events, their severity and frequency. The severity of adverse events was scored according to the National Cancer Institute (NCI) toxicity assessment criteria. Serum PSA and bone metabolism marker (pyridinoline cross-linked type I collagen carboxy terminal telopeptide: ICTP) levels were measured every 4 weeks during treatment. The blood concentration of ICTP was measured by a two-antibody radioimmunoassay (RIA) using an RIA kit of telopeptide ICTP (product of Espoo, Orion Diagnostica, Finland; provided by Chugai, Japan, Japan). The normal range of serum ICTP is 1.8-5.0 ng / mL [25]. Bone scans and abdominal computed tomography (CT) scans were performed every 3 months during the study. Findings of metastasis from bone scans were assessed by disease extent using percentage of positive parts in bone scan (% PABS) [26]. Clinical response was determined by both changes in PSA levels and changes in imaging studies in patients with measurable disease. A PSA response was defined as a 50% or greater reduction (PR) or normalization (CR) of PSA levels from baseline in PSA levels in two consecutive measurements at least 4 weeks apart. The time of PSA exacerbation was registered as the time when the PSA level increased by 25% or more from baseline for the first time in a row. Standard definitions were used for measurable and evaluable disease responses and exacerbations. For patients with two-dimensionally measurable disease, complete response (CR) was defined as the disappearance of the entire target lesion over at least 4 weeks; partial response (PR) was defined as two-dimensionally measurable disease. A reduction of ≧ 50% was defined, and a mild response was defined as a reduction between 25% and 50%. For bone metastasis response, CR was defined as the disappearance of the full positive range in the bone scan. For% PABS, PR was defined as a 50% or greater reduction, and exacerbation (PD) was defined as an increase in the number of positive sites, deterioration of existing lesions, or simultaneous observation of the two.

Statistical methodsNon-exacerbated survival is from the start of combination therapy to the time of exacerbation in patients with worsening disease, to the time of death in patients who die without exacerbation, or last in patients who have survived and have not progressed. It was defined as the time to contact. Cause-specific survival was defined as the time from the start of combination therapy to death due to disease. The Kaplan-Meier method was used to assess non-exacerbated survival and cause-specific survival.

Results Immunological response during combination therapy During combination therapy, peptide-specific CTL precursors and peptide-specific antibodies were measured at 11-week intervals for all 11 patients. Vaccinated peptides and immune responses are summarized in Table 2 below. During the combination therapy, all 11 patients who monitored the immune response had either a cellular or humoral response enhanced. An increase in peptide-specific CTL precursors was observed in 6 of 11 patients (cases 2, 5, 7, 8, 10, and 12), while induction of peptide-specific IgG was observed in 10 of 11 patients (case 3). 4, 5, 7, 8, 10, 11, 12, and 13). FIG. 1 shows the time course of both INF-γ production and the level of IgG specific for the peptide administered to each patient.

  Estramustine-induced immunosuppression was also analyzed in 10 out of 11 patients by measuring INF-γ production against PHA, EBV peptide and vaccinated peptide. In case 13, immune monitoring was not performed because too little PBMC was obtained for the assay. It has been suggested that responses to PHA, EBV peptide and vaccinated peptide (INF-γ production) are mediated by resting T cells, memory T cells and activated T cells, respectively. The monitoring results for each case are shown in FIG. Cases 2 and 3 were initially treated in combination with a full dose (560 mg / day) of estramustine phosphate, but immunological monitoring showed severe immunosuppression. This immunosuppression was restored by discontinuation of estramustine phosphate. When the peptide and half dose of estramustine phosphate were administered, there was no significant immunosuppression in any of the 8 cases (FIG. 2).

  The results of this study suggest the benefits of combined peptide vaccine administration and low-dose estramustine phosphate (280 mg / day) in patients with metastatic HRPC. This study demonstrated that in all patients with metastatic HRPC, cellular and humoral responses were well maintained during peptide vaccine administration and low-dose estramustine phosphate combination therapy. As a result of this study, it was shown that an increase in peptide-specific CTL precursors was observed in 6 of 11 patients, and induction of peptide-specific IgG was observed in 10 of 11 patients. When the peptide and low dose estramustine phosphate were administered, no significant immunosuppression was seen in any of 11 patients. Additional studies on a relatively large number of patients are recommended to confirm the results from this small study.

Clinical Response Table 3 summarizes the clinical response to peptide vaccine administration and oral estramustine phosphate combination. In 10 of 11 patients (91%), serum PSA levels dropped from baseline after treatment, among which 8 patients (73%) had reduced serum PSA levels ≧ 50%. FIG. 3 shows changes with time in the PSA level during the combination therapy period of each case. All eight patients who did not respond to prior estramustine phosphate chemotherapy had a PSA response. In 1 of 2 patients with measurable disease, CT showed a 44% reduction in lymph node metastasis (FIG. 4). The patient is still alive with a PSA reduction of ≧ 50% (case 12). Ten patients had bone metastases. There was no improvement in bone metastases, but 1 out of 10 patients with bone metastases showed a decrease in serum ICTP levels of ≧ 50%.

  Currently 3 patients died, all due to prostate cancer or metastasis. The median follow-up for all patients was 14 months and the range was 8-24 months. Median survival was not calculated. At 12 months, 64% of patients were alive.

  The overall response rate (73%) was defined as a ≧ 50% decrease in serum PSA levels, which was reported in previously reported immunotherapy (eg, interferon-α and interleukin-2 combination (31%) [32] or It is clearly higher than that seen in the Phase I / II report of infusions of dendritic cells (27%) [11, 12]) treated with peptides of prostate specific membrane antigen. In addition, this value is associated with chemotherapy clinical trials for combination therapy (eg, estramustine and paclitaxel (53%) [6], estramustine and docetaxel (62%) [7], and estramustine, paclitaxel and carboplatin). The response rate recently reported for the triple combination (67%) [8]) is comparable. For measurable disease, the overall response rate appears to be somewhat lower than reported in these chemotherapy regimens because the patients in this study had almost no measurable soft tissue disease . Most patients have reduced bone metabolism markers (ICTP), a proposed method for monitoring bone metastases in prostate cancer patients with bone metastases [33], but bone scans improve bone metastases Was not seen. The explanation for this discrepancy is that bone scanning is a less sensitive means, or the treatment period was too short to affect bone diseases that may be more resistant to therapy. might exist.

Toxicity All 13 patients were evaluated for toxicity. Toxicity reported in 13 treated patients is summarized in Table 4. This combination therapy was safely and well tolerated and had no significant side effects in most cases, but grade 3 arrhythmias and cerebral infarctions were observed in each case. One patient (case 4) developed a grade 3 arrhythmia that disappeared due to estramustine interruption. Patients hospitalized with grade 3 cerebral infarction after 14 months of combination therapy (Case 7) have been successfully treated with anticoagulants and are continuing combination therapy without any other significant toxicity. The most common toxicity was a skin reaction at the injection site of the vaccine in all cases. All 13 skin reactions were scored as grade 1 or 2 according to the National Cancer Institute's normal toxicity criteria. Seven patients complained of bone pain, four patients developed grade 2 hematuria, and three patients complained of fatigue. There was no blood, liver or kidney toxicity.

  The toxicity of the combination therapy reported here is weak, and this treatment is considered good for the treatment of elderly men (median age 71 years) with metastatic HRPC. The most common toxicity was a skin reaction in the local injection area. Importantly, there were no hematologic toxicities or neuropathies [4-8] reported to be dose limiting toxicities in estramustine-based or taxane-based chemotherapy. Common toxicities of estramustine treatment include nausea, vomiting, peripheral edema and vascular events [16]. This combination therapy has been found to be safe and well tolerated.

  Combination of patient-directed vaccination with low-dose estramustine phosphate resulted in a ≥50% reduction in serum PSA levels in 73% of patients studied and received mainly prior intensive care It was also less toxic in elderly patients with metastatic HRPC. Based on these preliminary findings, a larger phase II clinical trial was approved for this combination.

(Example 2)
Patient and Method A method substantially similar to Example 1 was adopted except for the following points.

Patients Between March 2002 and January 2003, phase I / II trials were conducted on 16 HLA-A24 positive metastatic HRPC patients. In 13 patients, if the condition worsened after at least the third peptide vaccine administration, the peptide vaccine administration and low-dose estramustine phosphate combination therapy were followed. The remaining three patients were only vaccinated because their condition worsened rapidly and resulted in death. Table 5 summarizes the clinical characteristics of 16 HRPC patients in this study.

Patient targeting peptide vaccination method This study used the following peptide 16 or derived from epithelial cancer-associated antigen and prostate-associated antigens: _{That, SART1 690-698 (EYRGFTQDF), SART2} 93-101 (DYSARWNEI), _{SART2 161-169 (AYDFLYNYL), SART2 899-907} (SYTRLFLIL), SART3 109-118 (VYDYNCHVDL), SART3 315-323 (AYIDFEMKI), Lck 208-216 (HYTNASDGL), Lck 486-487 (TFDYLRSVL), Lck 488 _{-497 (DYLRSVLEDF), ART1 170-179 (} EYCLKFTKL), prostatic acid phosphate _{(PAP) 213-221 (L CESVHNF), PSA 152-160 (CYASGWGSI)} , PSA 248-257 (HYRKWIKDTI), prostate specific membrane antigen _(PSMA) 624-632 _(TYSVSFDSL), multidrug resistance associated protein _{3 (MRP3) 503-511 (LYAWEPSFL)} , And MRP3 _1293-1302 (RYLTQETNKV). All of these peptides have the ability to induce HLA-A24-restricted and tumor-specific CTL activity in PBMC in cancer patients (15, 19, 20, 34-41). All patients were vaccinated with up to 4 peptides selected from 16 peptide candidates by pre-vaccine measurement.

Combination therapy Estramustine phosphate was administered orally at a dose of 140 mg capsules twice daily, avoiding severe immune suppression in combination therapy.

Clinical monitoring During this treatment, the Japanese version of the “Functional Evaluation of Cancer Therapy” (FACT-P) subscale, FACT-P (for prostate cancer) (42) was used to assess patient QOL before and after vaccination. Evaluation was made at the time of the 3rd, 6th and 12th vaccine administration. This FACT-P questionnaire shows physical health (7 items), social / family health (8 items), emotional health (6 items), functional health (7 items) and prostate cancer scale (12 items). It is composed of five factors. QOL results were assessed separately by percentage of each scale.

Results Immunological response during combination therapy. Peptide-specific CTL precursors were examined prior to peptide vaccine administration and were detectable in 14 of 16 patients, with a median number of positives per patient 1.5 peptides (range 0-4 peptides) (Table 6). Furthermore, anti-peptide IgG was detectable in 14 of 16 patients, and the median positive number per patient was 3 peptides (0-4 peptides). Up to 4 peptides were selected and injected into each patient, and the results of the selection are shown in Table 6. The most frequently selected peptide is PSA _248-257 (13/16), followed by PAP _213-221 (11/16), SART3 _109-118 (11/16), SART3 _315-323 (6/16). _{), PSA 152-160 (5/16),} Lck 488-497 (5/16), SART1 690-698 (3/16), SART2 93-101 (3/16), and _{Lck 208-216} (3 / 16) was selected. PSMA _624-628 and SART2 _161-169 were selected in each patient, but the remaining 5 peptides derived from SART2, Lck, ART1 and MRP3 were not selected in these patients. Increases in peptide-specific CTL precursors or peptide-specific IgG were observed in week 12 (peptide vaccine administration alone) in 10 of 14 patients or 7 of 14 patients and in week 24 (during combination therapy) ) Was observed in 6 of 8 patients or 10 of 12 patients. Representative results for peptide specific CTL precursors in PBMC are shown in FIG. FIG. 6 shows the gradual change in IgG levels specific for the peptide administered to each patient. DTH responses were observed in 4 out of 16 patients and are summarized in Table 6.

  When the peptide and half (280 mg / day) estramustine were administered, no significant immunosuppression was observed in most cases.

CLINICAL RESPONSE Two of the 16 patients with whom the tumor rapidly worsened prior to the combination therapy, the other patient withdrew consent for the combination therapy after serum PSA levels dropped ≥50% with vaccine alone did. The remaining 13 patients received vaccine and low-dose estramustine phosphate combination therapy to assess clinical response. In all 13 patients, serum PSA levels decreased from baseline after combination therapy, and 6 of 13 patients (46%) had a median 7.5 month (range) ≥50% decrease in serum PSA levels. 3 to 13 months). However, 13 of these patients showed no objective response to treatment.

  Patients with exacerbated disease have a short life expectancy and no hope of immediate healing, so reducing the physical condition and maintaining function are the primary goals of medical treatment [43]. In this study, the QOL results of 16 patients within the treatment period were determined before vaccine administration (16 patients) and the third vaccine administration (16 patients), the sixth vaccine administration (13 patients) and 12 At the time of the second vaccine administration (7 patients), the FACT-p Japanese version questionnaire was used for evaluation. FIG. 7 shows the average value of the scale percentage for each factor at the observation point. During the treatment period, QOL results did not decrease for all factors.

Toxicity All 16 patients were evaluated for general toxicity and the overall toxicity is shown in Table 7. The toxicity of the combination therapy reported here is tolerable and this treatment could be used to treat most metastatic HRPCs.

Example 3
As shown in Example 1, severe immunosuppression was observed at all doses (560 mg / day) of estramustine phosphate. The results of the combination therapy according to the present invention as seen from the dose of estramustine phosphate are summarized in Table 8. In combination therapy using estramustine phosphate at 280 mg / day (maximum non-immunosuppressive dose), patients with HRPC who showed an exacerbation of disease with peptide vaccination alone received increased immunological response and clinical An improved response was observed. These results support the beneficial effects of the combination of low dose estramustine and peptide vaccine administration.

Example 4
Table 9 also demonstrates that the combination therapy has superior effects compared to previous treatments. All cases did not respond to prior estramustine phosphate therapy and the condition worsened after vaccine administration alone (PD). Combination therapy improved clinical response to PR (partial response) or SD (stable condition) in most cases, and significantly extended the survival of HRPC patients (median survival: 25 months).

[Discussion]
Defining the expression of tumor antigens in various stages of prostate cancer is a crucial first step to select specific immunotherapy targets [28-30]. In the immunotherapy for HRPC patients, the method comprises administering up to four peptides among the vaccine candidates that have reacted in the pre-vaccine measurement following the pre-vaccine measurement of peptide-specific CTL precursors in the circulating blood of cancer patients. A new strategy was adopted (patient-directed vaccine administration). The results of previous phase I trials have shown that patient-directed vaccination is feasible, safe and immunologically effective, but the clinical response is generally limited [15] . In recent years it has been known that malignant transformation of cells is associated with altered expression and / or function of HLA class I and that this abnormality may provide tumor cells with a means to escape immune recognition. ing. In contrast to normal HLA class I expression in benign tissue, complete loss of HLA class I expression was reported in 34% of primary prostate cancer cells and 80% of prostate cancer cells with lymph node metastasis [31]. . Therefore, reduced expression of HLA class I antigens in prostate cancer may negatively affect the outcome of T cell-based immunotherapy by providing malignant tumor cells with a mechanism to escape recognition by T cells. is there. It is suggested that an additive anti-tumor effect can be achieved by the combined use of T cell-based immunotherapy and cytotoxic agents with minimal immunosuppression. In this study, PSA responses were also observed in patients who experienced exacerbations of disease prior to estramustine phosphate or peptide vaccine administration, a hypothesis that this combination therapy works with additive antitumor effects Support. However, the exact mechanism of this interaction is still unclear. Further research is needed on this mechanism.

[Literature]
1. Tannock IF, Osoba D, Stockler MR, Ernst DS, Neville AJ, Moore MJ, Armitage GR, Wilson JJ, Venner PM, Coppin CM, Murphy KC. Chemotherapy with mitxantrone plus prednisone or prednisone alone for symptomatic hormone-resistant prostate cancer : A Canadian randomized trial with palliative end points. J Clin Oncol 1996; 14: 1756-1764.
2. Kantoff PW, Halabi S, Conaway M, Picus J, Kirshner J, Hars V, Trump D, Winer EP, Vogelzang NJ. Hydrocortisone with or without mitoxantrone in men with hormone-refractory prostate cancer: Results of the cancer and leukemia group B 9182 study. J Clin Oncol 1999; 17: 2506-2513.
3. Hudes G, Einhorn L, Ross E, Balsham A, Loehrer P, Ramsey H, Sprandio J, Entmacher M, Dugan W, Ansari R, Monaco F, Hanna M, Roth B. Vinblastine versus vinblastine plus oral estramustine phosphate for patients with hormone-refractory prostate cancer: a Hoosier Oncology Group and Fox Chase Network phase III trial.J Clin Oncol 1999; 17: 3160-3166.
4. Smith DC, Esper P, Strawderman M, Redman B, Pienta KJ.Phase II trial of oral estramustine, oral etoposide, and intravenous paclitaxel in hormone-refractory prostate cancer.J Clin Oncol 1999; 17: 1664-1671.
5. Beer TM, Pierce WC, Lowe BA, Henner WD. Phase II study of weekly docetaxel in symptomatic androgen-independent prostate cancer. Ann Oncol 2001; 12: 1273-1279.
6. Hudes GR, Nathan F, Khater C, Haas N, Cornfield M, Giantonio B, Greenberg R, Gomella L, Litwin S, Ross E, Roethke S, McAleer C. Phase II trial of 96-hour paclitaxel plus oral estramustine phosphate in metastatic hormone-refractory prostate cancer. J Clin Oncol 1997; 15: 3156-3163.
7. Ellerhorst JA, Tu SM, Amato RJ, Finn L, Millikan RE, Pagliaro LC, Jackson A, Logothetis CJ. Et al: Phase II trial of alternating weekly chemohormonal therapy for patients with androgen-independent prostate cancer.Clin Cancer Res 1997 ; 3: 2371-2376.
8.Kelly WK, Curley T, Slovin S, Heller G, McCaffrey J, Bajorin D, Ciolino A, Regan K, Schwartz M, Kantoff P, George D, Oh W, Smith M, Kaufman D, Small EJ, Schwartz L, Larson S, Tong W, Scher H. Paclitaxel, estramustine phosphate, and carboplatin in patients with advanced prostate cancer.J Clin Oncol 2001; 19: 44-53.
9.van der Bruggen P, Traversari C, Chomez P, Lurquin C, De Plaen E, Van den Eynde B, Knuth A, Boon T. A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma. Science 1991; 254 : 16431647.
10. Kawakami Y, Eliyahu S, Sakaguchi K, Robbins PF, Rivoltini L, Yannelli JR, Appella E, Rosenberg SA.Identification of the immunodominant peptides of the MART-1 human melanoma antigen recognized by the majority of HLA-A2-restricted tumor infiltrating lymphocytes. J Exp Med 1994; 180: 347-352.
11. Tjoa BA, Simmons SJ, Bowes VA, Ragde H, Rogers M, Elgamal A, Kenny GM, Cobb OE, Ireton RC, Troychak MJ, Salgaller ML, Boynton AL, Murphy GP.Evaluation of phase I / II clinical trials in prostate cancer with dendritic cells and PSMA peptides. Prostate 1998; 36: 39-44.
12. Salgaller ML, Lodge PA, McLean JG, Tjoa BA, Loftus DJ, Ragde H, Kenny GM, Rogers M, Boynton AL, Murphy GP. Report of immune monitoring of prostate cancer patients undergoing T-cell therapy using dendritic cells pulsed with HLA-A2-specific peptides from prostate-specific membrane antigen (PSMA). Prostate 1998; 35: 144-151.
13. Mine T, Gouhara R, Hida N, Imai N, Azuma K, Rikimaru T, Katagiri K, Nishikori Misa, Sukehiro A, Nakagawa M, Yamada A, Aizawa H, Shirozu K, Itoh K, and Yamana H. Immunological evaluation of CTL precursor-oriented vaccine for advanced lung cancer patients.Cancer Sci 2003; 94: 548-556.
14. Tanaka S, Harada M, Mine T, Noguchi M, Gohara R, Azuma K, Yamada A, Morinaga A, Nishikori M, Katagiri K,, Itoh K, Yamana H and Hashimoto T .: Peptide vaccination for patients with melanoma and other types of cancers based on pre-existing peptide-specific cytotoxic T lymphocyte precursors in periphery.J Immunother (in press)
15.Noguchi M, Kobayashi K, Suetugu N, Tomiyasu K, Suekane S, Yamada A, Itoh K, Noda S. Induction of cellular and humoral immune responses to tumor cells and peptides in HLA-A24 positive hormone-refractory prostate cancer patients by peptide vaccination. Prostate (in press)
16. Perry CA and McTavish D. Estramustine phosphate sodium.A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic efficacy in prostate cancer.Drugs and Aging 1995; 7: 49-74.
17. Shichijo S, Nakao M, Imai Y, Takasu H, Kawamoto M, Niiya F, Yang D, Toh Y, Yamana H, Itoh K. A gene encoding antigenic peptides of human squamous cell carcinoma recognized by cytotoxic T lymphocytes. Med 1998; 187: 277-288.
18. Nakao M, Shichijo S, Imaizumi T, Inoue Y, Matsunaga K, Yamada A, Kikuchi M, Tsuda N, Ohta K, Takamori S, Yamana H, Fujita H, Itoh K. Identification of gene coding for a new squamous cell carcinoma antigen recognized by the CTLs. J Immunol 2000; 164: 2565-2574.
19. Yang D, Nakao M, Shichijo S, Sasatomi T, Takasu H, Matsumoto H, Mori K, Hayashi A, Yamana H, Shirouzu K, Itoh K. Identification of a gene coding for a protein possessing shared tumor epitopes capable of inducing HLA-A24-restructed cytotoxic T lymphocytes in cancer patients.Cancer Res 1999; 59: 4056-4063.
20. Gomi S, Nakao M, Niiya F, Imamura Y, Kawano K, Nishizaka S, Hayashi A, Sobao Y, Oizumi K, Itoh K. A cyclophilin B gene encodes antigenic epitopes recognized by HLA-A24-restricted and tumor-specific cytotoxic T lymphocytes. J Immunol 1999; 163: 4994-5004.
21. Maeda Y, Ito M, Harashima N, Nakatsura T, Hida N, Imai N, Sato Y, Shichijo S, Todo S, Itoh K. Cleavage and polyadenylation specificity factor (CPSF) -derived peptides can induce HLA-A2-restricted and tumor-specific CTLs in the majority of gastrointestinal cancer patients.Int J Cancer 2002; 99: 409-417.
22. Suzuki N, Maeda Y, Tanaka S, Hida N, Mine T, Yamamoto K, Oka M, Itoh K. Detection of peptide-specific cytotoxic T lymphocyte precursors used for specific immunotherapy of pancreatic cancer.Int J Cancer 2002; 98: 45-50.
23. Miyagi Y, Imai N, Sasatomi T, Yamada A, Mine T, Katagiri K, Nakagawa M, Muto A, Okouchi S, Isomoto H, Shirouzu K, Yamana H, Itoh K. Induction of cellular immune response to tumor cells and peptides in colorectal cancer patients by vaccination with SART3 peptides. Clin Cancer Res 2001; 7: 3950-3962.
24. Hida N, Maeda Y, Katagiri K, Takasu H, Harada M, Itoh K. A simple culture protocol to detect peptide-specific cytotoxic T lymphocyte precursors in circulation.Cancer Immunol Immunotherapy 2002; 51: 219-228.
25.Noguchi M and Noda S: Pyridinoline cross-linked carboxyterminal telopeptide of type I collagen as a useful marker for monitoring metastatic bone activity in men with prostate cancer.J Urol 2001; 166: 1106-1110.
26.Noguchi M, Kikuchi H, Ishibashi M, Noda S. Percentage of the positive area of bone metastasis is an independent predictor of thedisease death in advanced prostate cancer.Br J Cancer 2003; 88: 195-201.
27. Mitcell MS. Chemotherapy in combination with biomodulation: a 5 year experience with cyclophosphamide and IL-2.Semin Oncol 1992; 19 (suppl 2): 80-87.
28. Zhang S, Cordon-Cardo C, Zhang HS, Reuter VE, Adluri S, Hamilton WB, Lloyd KO, and Livingston PO. Selection of tumor antigens as targets for immune attack using immunohistochemistry. I. Focus on gangliosides. Int J Cancer 1997; 73: 42-49.
29. Zhang S, Zhang HS, Cordon-Cardo C, Reuter VE, Singhal AK, Lloyd KO, and Livingston PO. Selection of tumor antigens as targets for immune attack using immunohistochemistry. II. Blood group-related antigens. Int J Cancer 1997 ; 73: 50-56.
30. Zhang S, Zhang HS, Reuter VE, Slovin SF, Scher HI, Livingston PO. Expression of potential target antigens for immunotherapy on primary and metastatic prostate cancers.Clin Cancer Res 1998; 4: 295-302.
31. Blades RA, Keating PJ, McWilliam LJ, George NJ, Stern PL. Loss of HLA class I expression in prostate cancer: implications for immunotherapy. Urology 1995; 46: 681-687.
32.Maffezzini M, Simonato A, Fortis C. Salvage immunotherapy with subcutaneous recombination interleukin 2 (rIL-2) and alpha-interferon (A-INF) for stage D3 prostate carcinoma failing second-line hormonal treatment.Prostate 1996; 28: 282 -286.
33.Noguchi M, Yahara J, and Noda S: Serum levels of bone turnover markers parallel the results of bone scintigraphy in monitoring bone activity of prostate cancer. Urology 2003; 61: 993-998.
34.Noguchi M, Itoh K, Suekane S, Yao A, Suetsugu N, Katagiri K, Yamada A, Yamana H, and Noda S. Phase I trial of patient-oriented vaccination in HLA-A2 positive patients with metastatic hormone refractory prostate cancer Cancer Sci 2004; 95: 77-84.
35.Noguchi M, Itoh K, Suekane S, Morinaga A, Sukehiro A, Suetsugu N, Katagiri K, Yamada A, and Noda S. Immunological Monitoring during Combination of Patient-Oriented Peptide Vaccination and Estramustine Phosphate in Patients with Metastatic Hormone Refractory Prostate Cancer. Prostate 2004; 60: 32-45.
36. Inoue Y, Takaue Y, Takei M, Kato K, Kanai S, Harada Y, Tobisu K, Noguchi M, Kakizoe T, Itoh K, and Wakasugi H. Induction of tumor specific cytotoxic T lymphocytes in prostate cancer using prostatic acid phosphatase derived HLA-A2402 binding peptide. J Urol 2001; 166: 1508 -1513.
37. Harada M, Kobayashi K, Matsueda S, Nakagawa M, Noguchi M, and Itoh K. Prostate-specific antigen-derived epitopes capable of inducing cellular and humoral responses in HLA-A24 + prostate cancer patients.Prostate 2003; 57: 152- 159.
38. Kobayashi K, Noguchi M, Itoh K, and Harada M. Identification of a prostate-specific membrane antigen-derived peptide capable of eliciting both cellular and humoral immune responses in HLA-A24 + prostate cancer patients.Cancer Sci 2003; 94: 622 -627.
39. Harada M, Noguchi M, Itoh K. Target molecules in specific immunotherapy against prostate cancer.Int J Clin Oncol 2003; 8: 193-199.
40. Harashima N, Tanaka K, Sasatomi T, Shimizu K, Miyagi Y, Yamada A, Tamura M, Yamana H, Itoh K, and Shichijo S. Recognition of the Lck tyrosine kinase as a tumor antigen by cytotoxic T lymphocytes of cancer patients with distant metastases. Eur J Immunol 2000; 31: 323-332.
41. Kawano K, Gomi S, Tanaka K, Tsuda N, Kamura T, Itoh K, and Yamada A. Identification of a new endoplasmic reticulum-resident proteine recognized by HLA-A-24 restricted tumor infiltrating lymphocytes of lung cancer. 2000; 60: 3550-3558.
42. Esper PMoF, Chodak G., Sinner M, Cella D, and Pienta KJ. Measuring quality of life in men with prostate cancer using the functional assessment of cancer therapy-prostate instrument. Urology 1997; 50: 920-928.
43. Coons SJ, Kaplan RM. Assessing health-related quality-of-life: application to drug therapy. Clin Ther 1992; 14: 850-858.

FIG. 1A to FIG. 1J are graphs showing changes in INF-γ production specific to peptides administered in each case and IgG levels over time. In cases 2, 5, 7, 8, 10, and 12, an increase in peptide-specific CTL precursors was observed, and in cases 3, 4, 5, 7, 8, 10, 11, 12, and 13, induction of peptide-specific IgG Was observed. 2A to 2J are graphs showing the monitoring results of treatment-induced immunosuppression. Cases 2 and 3 showed severe immunosuppression, which was recovered by discontinuing estramustine phosphate full dose administration (560 mg / day). There was no immunosuppression in 8 cases receiving peptide and low dose estramustine phosphate (280 mg / day). 3A to 3K are graphs showing the time course of PSA levels during the study in 11 patients. Ten of the 11 patients (94%) had decreased serum PSA levels from baseline during combination therapy. FIG. 4 shows a CT image of case 12. A: Peraortic lymph node metastasis (arrow) was observed on the CT scan at the start of combination therapy. B: Recurrent CT scan 8 months after combination therapy reduced the size of lymph node metastasis by 44% (arrow). FIGS. 5A-5G are graphs showing representative results of peptide-specific CTL precursors in PBMC before and after vaccine administration. An increase in peptide-specific CTL precursors was observed with peptide vaccine administration alone and in combination therapy. High response (Ar): p ≦ 0.01 and 500 ≦ pure production (INF-γ level in response to corresponding peptide-INF-γ level in response to HIV peptide); A: Moderate response (A): p ≦ 0.05 and 50 ≦ pure production amount; B: p ≦ 0.05 and 25 ≦ pure production amount <50; C: 0.05 <p ≦ 0.1 and 50 ≦ pure production amount. FIGS. 6A to 6J are graphs showing step changes in administered peptide-specific IgG levels. The vertical line indicates OD and the horizontal line indicates serum dilution. An increase in peptide-specific IgG was observed with peptide vaccine administration alone and in combination therapy. Sc: Subcutaneous injection. Figures 7A through 7E are graphs showing the% QOL scale assessment during vaccine administration. n. s. : Not significant. QOL for all factors did not worsen during treatment.

Claims (5)

140-560 mg / day estramustine or a salt thereof to be administered to a prostate cancer patient together with a cancer antigen peptide that binds to the patient's HLA class I antigen and has a patient-specific CTL inducing ability A pharmaceutical composition for treating prostate cancer. The pharmaceutical composition according to claim 1, wherein the prostate cancer is hormone refractory prostate cancer. The pharmaceutical composition according to claim 1 or 2, wherein the patient is a patient who is not responsive to estramustine monotherapy. The pharmaceutical composition according to any one of claims 1 to 3, wherein the patient is a patient who is not responsive to cancer antigen peptide monotherapy. The pharmaceutical composition according to any one of claims 1 to 4, wherein the estramustine or a salt thereof is estramustine phosphate.

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